Received: 6 May 2020 Revised: 31 July 2020 Accepted: 2 August 2020

DOI: 10.1002/app.49832

ARTICLE

UV-shielding antimicrobial zein films blended with

essential oils for active food packaging

Manjot Kaur | Deenan Santhiya

Department of Applied Chemistry, Delhi

Technological University, Delhi, India
Correspondence
Dr Deenan Santhiya, Department of
Applied Chemistry, Delhi Technological
University, Bawana Road, Delhi-110
042, Delhi, India
Email: deenan.santhiya@dce.ac.in
Funding information
Delhi Technological University
Abstract
A comprehensive study on zein film blended with glycerol and essential oils
(EOs) is presented in this work. In particular, ultra violet (UV) shielding property
and antimicrobial efficacy of developed active zein films (ZF) are tested. The fabri-
cated films show zero transmittance in the UV region as compare to commercial
poly-film which shows 40%–80% of UV transmittance. Results show that films are
effective against spoilage microorganisms. The incorporation of EOs in ZF signifi-
cantly reduces the growth of microbes over fruit samples since day 3. Physical
interactions existing between EOs along with glycerol and zein provide consider-
able barrier properties. The glass transition temperature of the film comes out to
be 47.7 C having the tensile strength of 1.21 ± 0.05 MPa. TGA curves show major
weight loss in the range of 220–375 C. In conclusion, edible active ZF can be used
as healthy packages over food and drug to increase their shelf life.

KEYWORDS
biodegradable, biopolymers, packaging, proteins, renewable Polymers

1 | INTRODUCTION conventional packaging materials like HDPE (high-density

The Packaging industry is growing rapidly and nearly all
other industries are directly or indirectly depending on
it. When viewed in terms of packaging, the global share of
flexible plastic packaging is 42%.1 As reported by The Feder-
ation of Indian Chambers of Commerce and Industry FICCI
1/3rd of plastic products used by the packaging industry are
petroleum-based nonbiodegradable and environmentally
unfriendly. Out of the total global share, 24% of flexible
packaging is needed in the food packaging industry.2 These
plastics used for the packaging purpose are often discarded
and barely reprocessed.3 In this regard, in December 2018,
Great Britain's Royal Statistical Society conducted the study
and stated that only 9% of the plastic is being recycled.4 The
Abbreviations: EOs, essential oils; HDPE, high-density polyethylene;
PE, polyethylene; PET, polyethylene terephthalate; PP, polypropylene;
ZEO, zein film with glycerol and essential oil; ZF, zein film with
glycerol and without essential oil; ZP, pure zein protein.
polyethylene [PE]), polypropylene (PP), PE terephthalate
(PET), etc. though have excellent mechanical and barrier
properties5 but are unhealthy to the environment. They do
not have active properties, causing the growth of spoilage
and pathogenic microorganisms over the fruits and vegeta-
bles packaged into it.6–8 Traditional packaging also have
poor resistance to ultra violet (UV).9 They may undergo
photo-oxidation leading to polymeric structural degrada-
tion10,11 and release of free radicals.12 These free radicals
may enter into food materials packaged into it and the
human body. This can exert a detrimental effect on the food
quality as well as human health.13 To curb this problem and
to provide a shield against UV radiation, various UV
blockers, UV stabilizers like carbon black, titanium dioxide,
benzophenones, benzonitriles, etc. are added to plastics.
International agency for research on cancer has reported
titanium dioxide as a possible carcinogen to humans and
studies showed that the increase in lung tumors in rats is
associated with titanium oxide inhalation. 14

J Appl Polym Sci. 2020;e49832. wileyonlinelibrary.com/journal/app © 2020 Wiley Periodicals LLC 1 of 10

https://doi.org/10.1002/app.49832
2 of 10
Thus, with such a downside of synthetic polymers, its
associated health concerns, and the rapid increase in plastic
waste, extensive studies are conducted to produce active pack-
aging materials. For this purpose, various food-grade poly-
mers like proteins, polysaccharides, lipids15 have gathered
attraction and are being explored for the same purpose. Vari-
ous natural antimicrobial active agents such as essential oils
(EOs) containing eugenol, cinnamaldehyde16,17 are incorpo-
rated to make these active packaging. The usage of such bio-
based materials for antimicrobial packaging can reduce eco-
logical waste and eliminate undesirable changes in food qual-
ity.15,16 They can also act as a nutrient carrier.18 These edible
biodegradable polymers can be used as films and may be
directly applied onto the surface of the food product.19,20 In
food industries and medical sectors, the use of natural poly-
mers and food-grade additives have been steadily increasing
as coating materials over a drug, candies, fruits, etc.21,22
In this investigation, zein, a class of alcohol-soluble pro-
lamine hydrophobic storage protein found in corn attracts
the interest because of its film-forming ability, biodegrad-
ability, and biocompatibility.17,23 Food and Drug Adminis-
tration has approved it 'as one of the safe materials' for
pharmaceutical coatings because of its non-toxic nature. 24
Zein has been used in the food packaging industry as a
coating material due to its ability to provide a moisture bar-
rier. 23 But the classical brittleness and flexibility problems
of zein films (ZF)25 limit their use as a free-standing film.
Hence, to monitor limitations regarding flexibility,
active zein film packaging bearing UV shielding and anti-
microbial properties have been fabricated using EOs and
glycerol as cross-linker and plasticizer. 16,17,26 EOs are
biological substances produced by some plants as second-
ary metabolites and are distinguished by their volatile
nature.27 These EOs generally constitute terpenes and
terpenoids comprising as main group, aromatic and ali-
phatic constituents comprising the other group, all char-
acterized by low molecular weight. 28 They act as active
agents having germicidal antimicrobial property because of
the presence of major aromatic compounds (e.g., thymol,
carvacrol, and eugenol) present in concentrations of as
much as 85%27–29 and help in reducing, retarding, or even
inhibiting the growth of spoilage and pathogenic microor-
ganisms.30,31 Herein, cinnamon oil and clove oil are used,
having major constituents as cinnamaldehyde and acetyl
eugenol respectively. 29 These major constituents of chosen
EOs are not only expected to be natural cross-linkers but
also provide natural aroma 32 along with antimicrobial
properties to zein based food packaging material. More
importantly these aromatic compounds are expected to be
efficient in UV screening33 and attracted our attention
towards UV shielding edible packaging.
This work aims to the development of novel flexible
zein based food packaging material. Importantly, this
KAUR AND SANTHIYA
zein based film is expected to be functioning as a guard
against UV radiation without the addition of any kind of
UV blockers, stabilizers, etc. along with antimicrobial
property. The attempt of making these protein packaging
materials by simple solvent cast method using glycerol
and EOs is beneficial both to the ecosystem and human
health. Further, the fabrication of these films requires no
unhealthy chemicals and its processing produce no mal-
odorous toxic gases. The films formed have thoroughly
been characterized by standard characterization tech-
niques including their surface morphology. Also, the bar-
rier properties of the films are tested through a water
vapor permeability test (WVP) and oxygen transmission
rate (OTR) test along with their mechanical strength.
Further, the films are subjected to in-vitro antimicrobial
testing using the disk diffusion method.
2 | EXPERIMENTAL SECTION
2.1 | Materials
For the preparation of the film, zein protein in powder form
with 97%–99% purity (ZP, MW = 38 kDa), EOs (Cinnamon
oil, product number-W229105 and clove oil, product
number- C8392) and glycerol were purchased from Sigma-
Aldrich, Gallarate, Italy. Milli Q water was used for all the
experimental work and all other reagents used in this study
were of analytical reagent (AR) grade of high purity.
2.2 | Methods
2.2.1 | Fabrication of zein film
ZF were obtained by dissolving 0.5 g of zein protein (w/v)
in 80% ethanol with constant stirring at 45  C followed
by drop-wise addition of glycerol (Zein/glycerol weight
ratio 5:1) to the solution and stirred continuously for
30 min until a homogenous mixture was obtained
followed by cooling and casting onto PP petri-plates hav-
ing 5 cm diameter. So obtained ZF films were used as
control. Subsequently, 150 μl of the emulsion was pre-
pared using EOs (clove oil and cinnamon oil) and water
by mixing 45 μl of cinnamon oil and clove oil each and
60 μl of water. After the preparation, 120 μl of that emul-
sion was added simultaneously to the homogenous mix-
ture of zein/glycerol (as prepared for ZF) with constant
stirring for 30 min and cast after cooling the mixture. The
films obtained were named as ZEO (zein with essential
oil). These ZF and ZEO films obtained using the solvent
cast method were dried at 45 C in hot air oven overnight
for 18 h, and peeled off after complete drying. In this
KAUR AND SANTHIYA
way, zein based films with an average thickness of 0.296
± 0.015 mm were obtained using a precision micrometer.
2.3 | Characterization of the film
Fourier Transform Infrared Spectroscopy (FTIR): Dried
samples of films were ground and mixed thoroughly with
potassium bromide at the ratio of 1:100 and pelleted. The
IR spectra of pellets were then recorded using the NICO-
LET 380 FTIR operating in the range of 4000–400 cm−1
with a resolution of 4 cm−1.
XRD: XRD was performed on various ZF and pure
zein protein. Measurements were carried out with Rikagu
Miniflex-II operating at 20 kV and a current of 10 mA
 
using Cu-Kα radiations with scanning range 2θ of 5 –70

at 0.2 /min. A glass substrate was used to record the data.
Thermogravimetric analysis (TGA): The thermal stabil-
ity of the film samples was performed using TGA (TGA
4000, Perkin Elmer, USA). The samples were weighed
approximately 5 mg and heated from 0–600  C at a heating
rate of 10 C/min in an N2 gas flow of 50 ml/min.
Differential scanning calorimeter (DSC): Thermal
analysis was performed in a DSC (Perkin Elmer DSC
8000, USA). The sample of approximately 3 mg was
weighed inside an aluminium sample holder with a cover
that was hermetically sealed. For the experiment, heat

flow was kept at 3 C/min at a temperature range from 0
to 350 C with N2 as a purging gas at a flow rate of 50 ml/
min and with a cooling gas line at a flow rate of 2 ml/min,
which used an empty aluminium sample holder as a ref-
erence. Glass transition temperature (Tg) was determined
as a point of intersection of the tangent drawn at the
point of greatest slope on the transition curve with the
extrapolated baseline before the transition.
UV-visible spectroscopy (UV–Vis): To check the
shielding effect of the film against UV radiations, the
transmittance of PE (conventional packaging), ZF, and
ZEO was observed in the wavelength range 200 nm-
800 nm using UV–Vis Spectrophotometer (Cary 300 UV–
Vis-Agilent). Each film was assessed in five replicates.
Scanning electron microscopy (SEM): The surface
morphology of the film samples was studied with SEM
(ZESIS EVO MA15). The samples were gold coated and
then observed at an accelerating voltage of 5.0 kV at
10,000 magnifications.
Mechanical properties of the film (Breaking
Strength): Mechanical testing was done as per ASTM
D618-61(1990) standards using Instron Micro Tensile
Tester Model 5848, Singapore, with film dimension
(2.5 cm × 5 cm), load cell of 10 N, speed of elongation
(5 mm/min) film thickness 0.296 ± 0.015 mm. The tem-
perature of the laboratory was constantly maintained at
3 of 10
23–25  C and relative humidity (RH) was 50 ± 4% and
films were conditioned before testing. Film strips were
mounted on cardboard grips with initial grip separation
of 50 mm. Tensile strength was calculated by dividing the
maximum load that the film could withstand by the ini-
tial cross-section area of the strip. Elongation at break
(E %) was expressed by as.
E = 100 × (L1-Lo)/Lo..1
Water vapor permeability (WVP): Water vapor per-
meability was determined gravimetrically according to
standard method E 95–96 (ASTM, 1994) by water vapor
permeability tester (L-80-5000, Switzerland). The films
were inserted between the two chambers. The lower
chamber was filled with distilled deionized water and the
upper chamber was containing the sensor for controlling
the temperature and RH (25 C and 15% RH). Silica gel
was used as a desiccant for the adsorption of water
vapors. The moisture adsorbed by the silica gel was
detected periodically until a stationary state was reached.
For each measurement, three replications were made.
The final measurement was done after incubation for
2 days (48 h). The slope of the weight versus time plot
was divided by an effective film area to obtain the water
vapor transmission rate (WVTR). This was multiplied by
the film thickness and divided by the pressure difference
to obtain the water vapor permeability (WVP)34 and
water vapor permeability coefficient (WVPC) which indi-
cates the amount of water vapor that permeates per unit
of area and time in packaging material.
WVTR = slope/film area (g/m2).
WVP = WVTR× thickness/ΔP (g.mm/m2.kPa).
WVPC = ΔW × X / AΔT × ΔP (g.mm/m2.h.kPa) (2).
ΔW = weight gain by desiccant (g).
X = Film thickness (mm).
A = Area of exposed film (m2).
ΔT = incubation period (h).
ΔP = difference in partial pressure (k Pa).
Oxygen transmission rate (OTR): OTR was mea-
sured according to the ASTM F1927-14, (2014) stan-
dard using an oxygen permeation instrument
(OXTRAN model 2/20 Mocon, Minneapolis, MN,
USA). OTR of the film was determined under control

conditions (23 C and R.H = 0%). The film was placed
between two sides of the test chamber, one side
was exposed to carrier gas containing 98% N2 and 2%
H2 while the other side was exposed to the test gas
(pure O2), and the sensor present monitored the exit
port of the carrier gas measuring the amount of trans-
mission of oxygen. OTR was calculated according to
the following equation.35
OTR = V (O2)/ t.a (3)
V (O2) = measured volume of oxygen.
t = time required for reaching the stationary state (h).
4 of 10
a = exposed area of the film (m2).
In-vitro antimicrobial activity of films: (a) Disk diffu-
sion method: Antimicrobial activity of films (5 cm in
diameter) was conducted by using agar disc diffusion
method 36 against Gram-negative Escherichia coli ATCC
25922 and Gram-positive Staphylococcus aureus ATCC
29213 as test microorganisms. The overnight cultures of
bacteria were prepared in nutrient broth at 37  C. For
antimicrobial tests, agar plates were made, three for each
of the test microorganisms. After this, the bacterial inocu-
lums were spread over the agar plates. Disks from each
film were prepared under aseptic conditions of approxi-
mately 0.5 cm in diameter. They were placed over those
agar plates and were incubated at 37 C for 14–16 h and
the areas of the fully formed zones (ffz) were determined
by measuring the zone diameter with the digital caliper.
(b) Food sample testing: Fresh fruits samples of pear
were sliced (2.5 cm in length approximately), kept on
glass petri-plate having 5 cm diameter, and were covered
with the fabricated film (ZEO) and transparent PE poly-
film having 150 μm thickness (PE) to carry out the relative
study regarding the pace of spoilage of food. Also, one
petri-plate having the fruit sample was remained uncov-
ered. All the test samples were kept at standard tempera-
ture (25 C). With regular intervals from 1 day up to one-
week, samples were examined. After every regular interval,
food samples from each petri-plate were taken and diluted
in the nutrient broth. This followed the spreading of that
nutrient broth over the agar plates to see the growth of
micro-organisms over food sample which cannot be seen
with naked eyes. All experiments were done at ambient
conditions at a standard temperature at 25 C.
Statistical analysis: Data are expressed as mean ± SD.
Student's t test was used for the analysis of the test results
(least significant difference) at the significance level of
p-value <0.05.
3 | R ES U L T S A N D D I S C U S S I O N
At the macroscopic level, homogenous solvent-cast films
were obtained having a thickness of 0.296 ± 0.015 mm
and a diameter of 5 cm having see-through ability show-
ing the transparency of the film (Figure 1).
3.1 | Structural characterization
FTIR: FTIR spectra of films ZF and ZEO along with
native zein protein (ZP) are shown in Figure 2. For native
ZP, amide A band appearing between 2860 cm−1 and
3500 cm−1 belongs to the stretching of N-H and O-H
bond of the amino acids. The amide I band appearing at
KAUR AND SANTHIYA
1660 cm−1 belongs to the stretching of the carbonyl C=O
of the amide group of the peptide. Amide II band at
1536 cm−1 corresponds to the angular deformation vibra-
tion of the N-H bond and C-H stretching vibration. Also,
COŌ symmetrical stretching is observed at 1450 cm−1.
Moreover, the amide III band at 1246 cm−1 belongs to
the axial deformation vibration of the C-N bond and
C=O bending vibration. The observed FTIR peaks of ZP
are in good agreement with Yingling et al.37 In the case
of ZF, it is noteworthy that the absorption band appe-
aring at 1660 cm−1 (amide I) in ZP is shifted to lower
wavenumber at 1644 cm−1 indicating the existence of
hydrogen bonding between O-H of glycerol and C=O of
zein protein. Similarly, a shift (≈ 7 to 10 cm−1) in amide
II, amide III, and COŌ peaks of ZP is also observed
towards lower wavenumber for ZF. Fewer changes were
observed in the case of ZEO in the range of 2000–400
compared to ZF. Peak at 1039 cm−1 for ZF corresponds to
-C-O-H bond. It is pertinent to note the shortening of a
peak at 1038 cm−1 for ZEO might be indicating distortion
of -C-O-H bond. Interestingly, intermolecular hydrogen
bonding between cinnamaldehyde (-CHO) content of
EOs with –NH2 of protein in ZEO is evident by -NH
stretching at 3437 cm−1.38 In general, the observed peak
shifts towards lower wavenumbers confirm the existence
of inter- and intramolecular physical forces including
ionic, hydrogen bonding, and hydrophobic between zein
and glycerol molecules in ZF as well as zein and glycerol/
EOs in ZEO. A similar type of interaction is recently
reported between pectin and PEG. 39
XRD: X-ray diffraction patterns of various ZF along
with ZP are reported in Figure 3. XRD pattern of ZP indi-
cated two broad peaks centered at 9.2 and 20.3 . The
reported 9.2 and 20.3 peaks of ZP confirm the inter-
molecular intervals of 4.6 and 8.96 due to the average
distance of skeleton in the α-helical structure of protein
and distance of α-interhelix peaks in zein respec-
tively.40,41 Similarly, in the case of ZF also, these peaks
F I G U R E 1 Images of fabricated zein based films (a) ZF and
(b) ZEO. ZF, zein films; ZEO, zein film with EO [Color figure can
be viewed at wileyonlinelibrary.com]
KAUR AND SANTHIYA
F I G U R E 2 FTIR spectra of ZP, ZF, and ZEO. ZF, zein films;
ZEO, zein film with EO [Color figure can be viewed at
wileyonlinelibrary.com]
appeared with noticeable intensity reduction in addition
to lower 2θ shift for α-helix as well as higher 2θ shift for
α-interhelix peaks in comparison with the XRD pattern
of ZP. On the other hand, XRD spectrum ZEO film shows
an increase in the intensity of both the peaks with lower
2θ shift compared to ZF, but the intensity of both the
peaks are still less in comparison with ZP. These observa-
tions confirm that in general both helical structure, as
well as molecular accumulation of the protein matrix was
weakened in both the films ZF and ZEO. This is due to
the existence of inter- and intramolecular physical forces
between glycerol-native protein and glycerol/EO
molecules-native protein in ZF and ZEO films respec-
tively. Herein, XRD results very well corroborate with the
FTIR findings (Figure 2).
TGA: TGA was carried out to analyze the weight loss
of ZP and various films as a function of temperature. The
Thermal stability of ZP in its native form (powder), ZF,
and ZEO film was determined. The TGA curves are
shown in Figure 4(a). The observed thermal stability of
ZP was higher than ZF and ZEO due to its intact α-helix
structure as evident by XRD (Figure 3). The initial weight
loss of 3%–5% occurring in the range of 25 C to 120 C
was due to the evaporation of water.42 Between 120 C to
200 C, weight loss observed is accounted for the vapori-
zation of glycerol. This is due to the glycerol decomposi-
tion temperature, which is analyzed in isolation reported
around 210 C. 43 The major thermal degradation and
major weight loss of biopolymer of various samples
5 of 10
F I G U R E 3 XRD pattern of ZP, ZF, and ZEO with scanning
range 2θ of 5ο to 70ο at 0.2 /min. XRD, X-ray diffraction; ZF, zein
films; ZEO, zein film with EO [Color figure can be viewed at
wileyonlinelibrary.com]
(ZP, ZF, and ZEO) was observed from 220 C to 370 C
having 65.7% weight loss. The onset of major weight loss
for ZF starts from 187.3 C leading to 7.5% weight loss.
For ZEO, this temperature increased to 231.8 C having
weight loss near about 9%. The reported final weight loss
was 83%–85% at 574 C leading to the existence of 15%–
17% of residue and is in good agreement with various
researchers.40,42,44 It should be noted that, as revealed by
the derivative curve Figure 4(b), there was a slight shift
of degradation peak towards higher temperatures for the
ZEO film. Specifically, this peak was centered at 318  C
for ZF and ZEO it was at 326 C. These findings reveal
that degradation temperature of ZEO film is slightly
higher compared to ZF indicating the existence of addi-
tional crosslinking interaction of EO contents with zein
molecules as evident by FTIR and XRD (Figure 2 and 3).
DSC: Thermograms of the films (ZF and ZEO) pro-
duced by the casting method are portrayed in Figure 5.

The melting point (Tm) of ZF was obtained as 228.6 C,
Figure 5(a) which is lower than the ZEO (254.4 C),
Figure 5(b). Due to the amorphous nature of the zein pro-
tein, instead of a sharp melting peak, the broad range
was obtained showing the melting range of the tested
films. It is important to note that the increase in Tm of
ZEO is ascribed to more ordered zein molecules due to
the cross-linking interaction with the added EOs. This is
supported by the XRD pattern of ZEO film, which shows
an increase in the intensity of peaks correspond to α-helix
as well as α-interhelix compared to ZF (Figure 3). The
glass transition temperature of films is important as this
aspect limits the use of the material under required /
extreme temperature conditions and also provides
information regarding structural organizations like cross-
linking, crystalline nature, etc. Glass transition tempera-
ture (Tg) was determined by estimating the midpoint in
6 of 10 KAUR AND SANTHIYA

FIGURE 4
Thermogravimetric analysis of
ZP, ZF, and ZEO at a heating
rate of 10 C/min in N2 gas flow
of 50 ml/min (a) TGA (b) dTGA
thermograms. TGA,
thermogravimetric analysis; ZF,
zein films; ZEO, zein film with
EO; ZP, zein protein [Color
figure can be viewed at
wileyonlinelibrary.com]

F I G U R E 5 DSC
thermograms of ZF and ZEO
films at heat flow 3 C/min and
with N2 as a purging gas at a
flow rate of 50 ml/min (a),
(b) Tm, (c), and (d) Tg. DSC,
differential scanning
calorimeter; ZF, zein films;
ZEO, zein film with EO [Color
figure can be viewed at
wileyonlinelibrary.com]

the proximity of the change in specific heat (ΔCp). Lines
tangential to Cpsolid to Cpliquid and transient in between
were drawn and the intersection between Cpsolid, Cpliquid,
and transient was defined and designated as Tg.45 The
glass transition temperature of ZEO came out to be
47.7 C, Figure 5(d) which is much lower to the ZF
(77.6 C), Figure 5(c), similar to which is reported by
other researchers. 40,42 It is thought that depression in Tg
could be attributed to the additional crosslinking pro-
vided by acetyl eugenol present in clove oil as well as cin-
namaldehyde content of cinnamon oil with zein
molecule. The decrease in Tg is due to the plasticization
of zein protein fibers by the EOs in ZEO film. ZEO con-
tains both glycerol and EOs leading to larger amount of
plasticizer and lowering of Tg.46
UV Visible Spectroscopy: UV–Visible spectroscopy
was conducted to check the ability of the fabricated film
to shield UV radiation and comparing it with conven-
tional packaging (PE poly-film). It was evident from the
graph in Figure 6 that PE did not absorb the wavelength
in the UV region leading to transmission of UV radia-
tions from 40% to 80% in the range of 200–400 nm. The
UV transmittance of ZEO and ZF is almost zero increas-
ing up to 1.56% at 432 nm for ZF and 0.69% at 446 nm for
ZEO film which proves it to be UV protecting. This is
attributed to the protein nature of the film. 47 Also, ZEO
has a little more shielding effect against UV radiations
because of the presence of aromatic compounds present
in the EOs that have been incorporated into it. 48 If we
compare our data with other biobased films as quoted by
KAUR AND SANTHIYA
Vilela C. et al. chitosan/ellagic acid films, ZEO films
show remarkable UV protection behavior as compare to
chitosan/ellagic films that showed approximately 20%–
30% of UV transmittance. 49 The addition of EOs consid-
erably decreased the UV transmittance which is in agree-
ment with Martin et al. Where it was quoted that
protection against UV is increased by increasing the
active components.48,50 Thus suggesting ZEO films to be
good UV absorbers and can help in preventing photo-
oxidation of the photo-sensitive food or pharmaceuticals.
F I G U R E 6 UV transmittance of PE, ZF, and ZEO. ZF, Zein
films; ZEO, Zein film with EO [Color figure can be viewed at
wileyonlinelibrary.com]
F I G U R E 7 SEM
micrographs of films at an
accelerating voltage of 5.0 kV
(a) 10,000×, (b)1000× - ZF, (c)
10,000× and (d) 1000× - ZEO.
SEM, scanning electron
microscopy; ZF, Zein films; .
ZEO, Zein film with EO
7 of 10
3.2 | Morphological determination
by SEM
The microstructure of ZF and ZEO films was observed
using SEM depicted in Figure 7. From the images, it was
observed that ZF Figure 7(a) at magnification 10,000×
and Figure 7(b) at 1000× magnification has a rough
bumpy surface as a result of pure zein and glycerol asso-
ciation which is in agreement with Khalil et al.51 and as
per Ming Zeng. 52 With the addition of EO, it leads to the
formation of a smoother ZEO surface as in Figure 7(c) at
10,000× and Figure 7(d) at 1000× having homogenous
nature 42 due to the interaction between EO and protein
fibers. It seemed that acetyl eugenol and cinnamaldehyde
contents of EOs have good affinity with zein protein
driven by physical interactions and increasing the film
networking making it homogenous and smooth.46
Tensile testing: In Table 1, it is depicted that ZF
has shown poor mechanical properties having a tensile
strength of 0.94 ± 0.047 MPa and elongation at break (E%)
at 3.72 ± 0.015%. After the addition of EOs, a higher value
of tensile strength 1.21 ± 0.054 MPa, E% 4.93 ± 0.24% was
obtained due to the additional crosslinking provided by EO
contents as explained by FTIR and XRD findings
(Figures 2 and 3) and agrees with earlier reported work. 41
In detail, cinnamaldehyde and acetyl eugenol (present in
EOs) interact with zein network along with glycerol
through physical forces. This leads to stabilization and
alignment of protein fibers in a compact form which could
be the reason for the enhancement of the mechanical
8 of 10 KAUR AND SANTHIYA

properties of the ZEO film. The breaking force of the ZEO glycerol entrapped in the polymer matrix before drying
of width approximately 25 mm also came out to be 46.0 and is in agreement with the data quoted by Tilminlioglu
± 2.07 N slightly greater that ZF (38.4 ± 1.53 N). The data et al. 55 having GLY/50 (Plasticizer type/Concentration)
showed no significant difference.
Water vapor permeability of fabricated films: It is well
known that water vapor barrier property of packaging
material is an important factor in determining the shelf
life of food products. Water vapor permeability values
(g mm/kPa.h.m2) of ZEO and ZF were calculated. From
the calculation, as indicated in Table 2, water vapor per-
meability for ZF is higher (5.47 ± 0.27 g.mm/kPa.h.m2)
as compare to ZEO (4.31 ± 0.21 g.mm/kPa.h.m2) show-
ing no such significant difference in data and found in
agreement with Vahedikia et al.41 Hydrophobic zein pro-
teins bonds with EO contents via hydrogen bonding and
thus impeding the bond formation with water molecules
and reducing the absorption of water.53 Due to the pres-
ence of EOs, permeability is influenced by the hydropho-
bic nature of the EO.41
OTR: Shelf life of food products is enhanced when F I G U R E 8 Disk diffusion test for antimicrobial activity at
the oxygen barrier property of the film is high. TheOTR 37 C against- lane (1) (left to right) Escherichia coli (a) blank,
of both films was measured at 23 C and 0% RH and (b) ZF and (c) ZEO. Lane (2) (left to right) Staphylococcus aureus
results are shown in Table 2. Zein film has a lower value (a) blank, (b) ZF and (c) ZEO. ZF, zein films; . ZEO, zein film
of OTR and shows greater gas barrier property than lower with EO [Color figure can be viewed at wileyonlinelibrary.com]
density PE (LDPE), high-density PE (HDPE), Polystyrene
(PS), Poly Vinyl Chloride (PVC).46,54 In our study, the
OTR value of ZEO came out to be (0.0705 ± 0.0035 cm3/
m2/day) which is better than the OTR value of ZF
(0.0965 ± 0.0048 cm3/m2/day). The observed trend could
be due to homogenizing effect of EOs46 along with

T A B L E 1 Mechanical properties of zein based films as per

ASTM D618-61(1990) standards

Film Tensile strength (MPa) Elongation at break (%)

ZF 0.94 ± 0.04 3.72 ± 0.01
ZEO 1.21 ± 0.05 4.93 ± 0.24

Note: Values are mean ± standard deviation and they were not sta-
tistically different (p < 0.05).
Abbreviations: ZEO, Zein film with EO; ZF, zein film.

T A B L E 2 Barrier properties of zein based films -water vapor

permeability (ASTM E 95–96, 1994) and oxygen transmission rate
(ASTM F1927-14, 2014)

Film WVP (g.mm/kPa.h.m2) OTR (cm3/m2/day)

ZF 5.47 ± 0.27 0.0965 ± 0.0048
ZEO 4.31 ± 0.21 0.0705 ± 0.0035

Note: Values are mean ± standard deviation and they were not sta- FIGURE 9 Antimicrobial testing over food sample at 25 C
tistically different (p < 0.05). without cover (w/o) and covered with PE and ZEO (left to right)
Abbreviations: ZF, zein film, ZEO, zein film with EO; OTR, oxygen respectively on days 1, 3, 5, and 7. ZEO, Zein film with EO [Color
transmission rate; WVP, water vapor permeability. figure can be viewed at wileyonlinelibrary.com]
KAUR AND SANTHIYA
and 95/5 (Ethanol % (v/v)/Corn-zein(w/v) concentration.
The data showed no significant difference.
3.3 | In-vitro studies
The antimicrobial activity of the films was performed
using the disk diffusion method. It can be observed from
Figure 8 (1) (c) and (2) (c) that ZEO showed pronounced
antibacterial activity against both E. coli and S. aureus by
showing a significant zone of inhibition approximately
20 ± 1 mm in diameter. The antimicrobial activity of this
active packaging is credited to the presence of EO.51 ZF
as shown in Figure 8 (1) (b) and (2) (b), did not show any
antimicrobial activity. After observing these results, ZEO
film was tested as packaging material over a food sample,
and results were compared with conventional packaging
material PE poly-film. From Figure 9, we can see that the
fruit samples were kept covered by ZEO film, PE, and
one without any covering. The film testing over the food
sample showed prominent results for the ZEO films
exhibiting delayed deterioration of the food sample pack-
aged into it. This was made evident by culturing the food
samples over the agar plates to see any growth of micro-
organisms. From Figure 10 it can be observed that on 3rd
day, there was no microbial growth in ZEO film samples
and after 7th day of examination of food samples, one
bacterial colony was observed in ZEO film packaging
unlike PE poly-film and uncovered food sample where
the bacterial growth can be seen from 3rd day itself. All
these results indicate ZEO as a potential packaging mate-
rial and EOs as natural ingredients in active packaging
F I G U R E 1 0 Microbial growth over food sample without
cover (w/o) and covered with PE and ZEO (left to right)
respectively on days 3 and 7 at 25 C. ZEO, Zein film with EO
[Color figure can be viewed at wileyonlinelibrary.com]
9 of 10
material to provide antimicrobial characteristics to the
packaging films.
4 | CONCLUSION
This manuscript successfully presents a simple fabrication
of zein film incorporated with EOs with zero UV transmit-
tance and antimicrobial property by forming a strong zone
of inhibition. Due to added EOs in addition to glycerol,
there increases the thermal and mechanical stability of
ZEO as compare to ZF. Water vapor permeability and oxy-
gen transmission rate on these active films showed sub-
stantially significant barrier properties and increases the
shelf life of the packaged food. Lower mechanical resis-
tance of these edible films as compare to conventional
poly-film directs their usage as coating materials over food,
drugs, etc. and for unit packaging and laminates. These
findings promise not only cost-effective and healthy food
packages but also can decrease the usage of toxic plastics.
ACKNOWLEDGMENT
The authors would like to thank Technical Education
Quality Improvement Programme (TEQIP) Phase-III,
Delhi Technological University, New Delhi for financial
support, and also acknowledge Vikas Kumar Chanchal
for experimental help.
CONFLICT OF INTEREST
The authors declare no potential conflict of interest.
ORCID
Deenan Santhiya https://orcid.org/0000-0002-0701-
2456
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How to cite this article: Kaur M, Santhiya D.
UV-shielding antimicrobial zein films blended with
essential oils for active food packaging. J Appl
Polym Sci. 2020;e49832. https://doi.org/10.1002/
app.49832